Gene targeting by homologous recombination (HR) in mouse embryonic stem (ES) cells provides a powerful technique to investigate gene function and generate human disease models. But similar technique has not been achieved in other research systems, e.g., rat, because of difficulties of generating and/or maintaining ES cells. However, in the past few years zinc-finger nucleases (ZFNs)-based technique has been developed and used for the generation of gene-targeting organisms with high efficiency. Double strand DNA breaks (DSB) generated by ZFNs stimulates cellular DNA repair, both by non-homologous end-joining (NHEJ) and HR pathway. The generation of gene targeting rat through embryo injection of ZFNs and NHEJ pathway has also been achieved, but not by HR pathway, while the latter one has wider applications. The goal of this proposal is to optimize conditions for modification of rat genome through HR by embryos injection of ZFNs. We hypothesize that the recruitment of donor DNA to DSB site will increase the efficiency of HR. Rat Tyr gene is selected as a target. Two donor DNAs, containing mouse Tyr gene and ERT2CreERT2 ubiquitous or HcRED germ cell-specific expression cassette, will be constructed to replace partial rat Tyr gene. ZFNs and donor DNAs will be first tested in rat fibroblasts, then injected into rat embryos by pronuclear injection to optimize the procedure. Successful knockout/knockin of rat Tyr gene by HR will have coat color change, but also these rats will ubiquitously express ERT2CreERT2 or express HcRED in germ cells. This technique should accelerate generation of rat models for biomedical research. PUBLIC HEALTH RELEVANCE: Transgenic animal models are an important tool for human diseases study and drug development. The overall goal of this project is to develop an innovative strategy (using zinc finger nucleases and donor DNA) for homologous recombination in rat embryos. This method will enable "knockout" or "knockin" approaches for studying gene function.